DE2735480A1 - PROCEDURE FOR THE SELECTIVE INACTIVATION OF PROTEOLYTIC ENZYMACTIVITY IN A BACTERIAL ALPHA AMYLASE ENZYME PREPARATION - Google Patents

Description

MÜLLEU-ISOITE DEUl'EL SCIIO-V ¹ II-KTWL

PAT E N TA NWiLT E

DR. WOLFGANG MÜLLER-BORE

(PATENT ADVERTISER FROM 1927-1975)

DH. PAUL DEUFEL. DIPL-CHEM. DR ALFRED SCHÖN. DIPL.-CHEM. WERNER HERTEL, DIPL.-PHYS.

C 3016

CPC International, Inc.
International Plaza, Englewood Cliffs
New Jersey 07632

Method for the selective inactivation of the proteolytic enzyme activity in a bacterial
O-amylase enzyme preparation

709886/1012

H MI'SCIIEX SlI · SI ElIKKTSTIt. + · POSTl-ACIl 8 «O7iO · K.VUKI .: MOKUOl 'AT · Ti: f .. (OMi)) 17400.1 ■ TIlLEX .1-JiJS

description

The invention relates to, more particularly, the selective inactivation of one enzyme in the presence of another enzyme the selective inactivation of the proteolytic enzyme activity associated with the OL amylase activity is present in crude bacterial d-amylase enzyme preparations.

The dL-amylase is an enzyme preparation that for Vex'dauing or liquefying starch materials as an initial stage making a number of sweeteners, such as dextrose, levulose, maltose and corn syrups with a high De: ctro3equivalent (D. E.), is used. The cL-amylase hydrolyzes starch molecules by dividing them into a multitude broken down from fragments with a lower molecular weight. Mose products are used to make the desired End product then with one or more additional Enzyme preparations such as B. Glucoamylase, / 3 -aciylase and glucose isomerase. Alternatively, a large number of these enzyme preparations can be put into one slurry at the same time of the starch material to make the sweetening materials directly.

Ck amylase enzyme preparations can be made from a large number of Sources are obtained. It is supreme from an economic and especially from a commercial point of view advantageous to use ö - atnylases, which are formed by bacteria will. Certain bacterial organisms, such as Bacillus subbilis, Bacillus licneriiformis and the like are described in cultured in large containers, the cells are then destroyed and the enzyme preparation is separated from the broth and

709886/1012

cleaned. This "cleaned" ct. - Contains amylase enzyme preparation however, there are still certain contaminants that are difficult and costly to remove. Such a detrimental pollution is known as protease or proteolytic enzyme. This biologically active material can with various existing in the starch materials proteinaceous materials react to form Hydrolysates containing peptides, polypeptides and amino acids.

The presence of the protease enzyme contaminant in the oil amylase is deleterious in hydrolyzing the starch-containing material. In general, different types of starch materials contain different amounts of proteinaceous materials. For example, a purified corn starch material can contain only 0.3% protein, a dry milled product, such as corn grits, can contain up to about 8 or 10 % protein, and unpurified starch materials called mill starches can contain up to 20% proteinaceous materials contain. When the starch material is hydrolyzed using the oil-amylase enzyme preparation, the presence of the protease activity in the preparation leads to a breakdown of the protein material which is to be hydrolyzed at the same time as the starch. This leads to a loss of economically valuable protein products. In addition, these undesirable by-products must be removed from the final sweetener product, which creates additional costs.

It is economically desirable to produce the starch hydrolysates to use the cheapest possible starting material. Therefore, a mill strength from which only the fibers

709886/1012

and seedlings have been removed is preferred to a purified starch which is low in protein. When using a cx-amylase enzyme preparation that Having significant amounts of protease activity requires relatively expensive purified starch materials to use because of their low protein content. The application of such enzyme preparations to less purified starch materials would lead to the formation of excess protein hydrolyzate materials which subsequent use and conversion of the starch hydrolyzate would interfere.

When the CL amylase enzyme preparation is essentially free from of proteolytic enzyme activity, the proteins contained in the starch material will be hydrolyzed should not be hydrolyzed to any appreciable degree, but remain with the insoluble material and can be effective Way filtered and separated (recovered).

It is not essential that all of the proteolytic enzyme activity be removed or inactivated in the oil amylase enzyme preparation will. Although it is preferred that this proteolytic activity be reduced to as low a level as possible is found that if the ratio of proteolytic enzyme activity (U / g) to amylolytic enzyme activity (U / g), d. H. the P / o (.- ratio, is less than about 2, of the enzyme preparation for the purposes of the present Invention works in a satisfactory manner. It is particularly preferred that this ratio be less than is about 0.8. As used herein, "protease-free cC-amylase" means an o (-amylase enzyme preparation to understand in which this ratio is less than about 2

709886/1012

amounts to.

The protease-free <x -amylases of the invention are suitable for the production of starch hydrolysates from a wide variety of starchy materials. These can be obtained from any source, for example from corn, wheat, rice, Potatoes and grain orghum. Waxy materials can also be used. In the US are out Corn-derived starchy materials are generally preferred for economic reasons, while in other parts In the world, starchy materials derived from cereals (wheat) and rice are preferred.

The starchy material can be any of a variety of partially purified products; H. around dry materials, such as corn grits (other examples include corn sludge, corn meal, Wheat flour and the like), or wet products, such as. B. Mill starch, and more purified products from which some of the gluten has been removed, act. The term "mill strength" as used herein means any of the numerous partially purified starch slurries within the range of a very crude product from which only the fibers and seedlings have been removed to a purified material that is less than about 0.3% proteinaceous Contains materials. "Light mill strength" is to be understood as the starch slurry, as it is from the Pasern and seedling separators is obtained while under "heavy mill strength" means a similar material from which some of the water has been removed. It pure starches can also be used.

709886/1012

The protease-free ol-amylases of the invention are suitable for the production of starch hydrolysates according to conventional processes. They are particularly suitable for use in Process in which the remaining, unconverted starch remains in its granular form. Such procedures are in U.S. Patents 3,922 196, 3,922 197, 3,922 198, 3 922 199, 3 922 200 and 3 922 201.

In the case of the granular starch processes, at least the initial solubilization is used the starchy material at a relatively low temperature; d. H. below the respective Gelatinization temperature of the respective starch contained in the starch material. At these temperatures protease activity poses a particular problem because it does not inactivate to any significant degree will. When using conventional procedures in which higher temperatures are used, the proteolytic Enzymes inactivated to a partial degree during use.

A number of methods have been proposed for removing, reducing, or inactivating the proteolytic or other enzyme contaminants in cx-amylase and other enzyme preparations. Thus, the inactivation of liquefaction enzymes in an amyloglucosidase preparation is described, for example by heating to about 100 ⁰ C (210 ° F) in U.S. Patent 3,249,512; US Pat. No. 2,683,682 describes the differential inactivation of OL amylase and proteinase mixtures by adjusting the pH of the mixture and heating to selectively inactivate one of the enzymes; U.S. Patent 3,912,590 states that the protease is in one of Bacillus

709886/1012

licheniformis derived ol-amylase can be inactivated during starch liquefaction if at least the initial part of the process is carried out at a temperature of 100 to ^{115 C} ; U.S. Patent 3,303,102 describes the use of a starch hydrolyzate to buffer a fungi amylase preparation from inactivation during acid inactivation of transglucosidase. Japanese Patent 16 696/62 also states that proteolytic enzyme solutions can be stabilized by a combination of polyhydroxy alcohols and calcium salts.

It has now been found that in the crude bacterial oil amylase enzyme preparation the protease activity can be decreased by heating the crude preparation to a predetermined one Temperature for a predetermined period of time to inactivate the protease. Before this heat treatment is the crude bacterial dL-amylase enzyme preparation with a material mixed, which is able to protect the dL-amylase against inactivation. A preferred protective material is a carbohydrate substrate material such as corn syrup. Alternatively, as a further preferred embodiment in cL-amylase preparations, derived from Bacillus licheniformis, which are particularly heat-resistant, the proteolytic activity can be reduced by a similar heat treatment, but using calcium ions as a protective material.

Below the inactivation of the enzyme preparations is the decrease the defined activity of the protease enzyme to be understood to be less than the ratio given above. the cL-amylase activity of the enzyme preparation is determined according to the U.S. Patent 3,922,199.

709886/1012

One unit of protease enzyme activity is defined as the amount of enzyme that forms trichloroacetic acid-soluble fragments, as determined in the test procedure given below result in a blue color corresponding to 0.5 / ^ g of tyrosine.

A standard casein solution is prepared by dissolving 2.00 g of casein in 20.0 ml of 1.0 η NaOH. There are 50 ml of water added and the pH is adjusted with dilute phosphoric acid (1 part of 85% acid to 3 parts of water, based on the volume) adjusted to 6.3. It becomes water for replenishment added to 100 ml. A tyrosine stock solution is prepared by dissolving 250.0 mg of 1-tyrosine in 0.2 η HCl for the preparation of 500 ml solution. This solution includes pro ml 500 ^ g tyrosine. A Folin Phenol reagent solution is prepared by diluting 100 ml of phenolic reagent (2N Folin and Ciocalteau, Fisher Scientific Co.) with 300 ml of water. A standard curve is prepared as follows:

1.0, 2.0, 3.0, 4-, O, 5.0 and 6.0 ml of the tyrosine stock solution are pipetted into each of six 50 ml volumetric flasks and diluted to the mark with 0.2N HCl. The solutions obtained contain 10, 20, 30, 40, 50 and 60 μg of tyrosine / ml. 3.0 ml of each solution are placed in a 25 x 150 mm test tube pipetted in. A blank sample is prepared which contains 3.0 ml of 0.2 η HCl. In each test tube, 15.0 ml 0.4 M NagCO, and 3.0 ml Folin phenol reagent are pipetted in and mixed with a vortex mixer. The whole thing is placed in a 37 ° C water bath for minutes to let the color change to develop. The absorbance of each sample in relation to distilled water is then measured in a 1 cm cuvette nm determined. The absorbance of the blank is determined by the absorbance subtracted from each sample and the corrected absorbance

709886/1012

is plotted against the amount of tyrosine in ^ ug / ml. The absorbance values should be within the range of 0.10 to 0.60.

The following protease test procedure is performed: By adding a known amount by weight of the enzyme preparation to a 500 ml volumetric flask and dilute to the mark with a 0.1 w / v% calcium acetate solution prepared an enzyme solution containing 20 to 100 U / ml protease. 1.0 ml of enzyme solution is added to each of two 15 ml centrifuge tubes in a water bath at a constant temperature of 37 ° C (one tube is for the sample, the other used for a blank sample). Allow to equilibrate for 5 minutes. Using blow-out pipettes 1.0 ml of a casein solution is added to the sample and 2.0 ml of 0.4 M trichloroacetic acid are added to the blank sample and mix using a vortex mixer. After a reaction time of exactly 10 minutes, 2.0 ml of 0.4 M are pipetted Trichloroacetic acid in the sample and 1.0 ml casein solution in the blank.

Mix each sample as indicated above and continue incubation for an additional 20 minutes at 37 ° C. After removing the samples from the bath, centrifugation is carried out immediately for 10 minutes at 2000 rpm. The supernatant liquid is decanted into a 25 x 150 mm test tube and mixed thoroughly. 2.0 ml of each supernatant liquid is pipetted into a 25 x 150 mm test tube. 10.0 ml of 0.4 M Na ₂ CO ₅ and 2.0 ml of Folin phenol reagent are pipetted into each test tube and mixed. The test tubes are placed in a 37 ° C water bath and incubated for exactly 20 minutes to develop color. It is related to the absorbance

709886/1012

- 33-- Ju

determined on distilled water in a 1 cm cuvette at 660 nm. After subtracting the absorbance of the blank from the absorbance of the sample, the net absorbance and are obtained the apparent tyrosine concentration is determined from the standard curve. The protease activity is then calculated as follows: Protease activity (U / g) = apparent tyrosine concentration (/ Ug / ral) χ 2 χ 2 (dilution factor).

Any ct-amylase can be used in the first embodiment of the invention. These preparations come from a variety of materials. These are common commercial products that are sold under a wide variety of product names in the trade. Particularly preferred for the practice of the invention are α-amylase enzyme preparations with a relatively high heat resistance, e.g. B. those preparations which are sold under the trademark Thermamyl, a product of Novo Terapeutisk Laboratorium, Copenhagen, Denmark. These enzyme preparations are derived from certain strains of Bacillus licheniformis, as generally described in British Patent 1,296,839, which have a relatively high heat resistance. Other useful enzyme preparations are those derived from Bacillus subtilise, ζ. B. those sold under the trademarks Rapidase and Tenase.

The bacterial α-amylase enzyme preparation is first treated with a Protective material mixed. Suitable protective materials are generally carbohydrate-based materials that reduce heat reduce the α-amylase enzyme activity in the preparation can. However, these same protective materials generally do not protect the proteolytic enzyme activity in

709886/1012

the same dimensions as for the a-amylase. In general The protective materials that are preferred according to the invention are materials that can be attacked by α-amylase and can thus protect the active center, such as B. starch hydrolyzate materials having a D.E. within the range of up to about 50, e.g. B. a hydrolyzed, solid cereal product having a D.E. of 9 to 11 (e.g. a maltodextrin) sold under the trademark Mor-Rex by from CPC International Inc, Englewood Cliffs, New Jersey / USA, or a corn syrup with a D.E. of 42, which is also distributed by CFC International Inc. It can also be used with dextrose as a protective material some decrease in effectiveness. Correspondingly, the protective effect is also lower when the starch hydrolyzate an end. of much more than 50 has. These protective materials decrease when used in a volume ratio of protective material to enzyme within the range of about 2: 1 to about 1: 2, the rate of heat inactivation of the α-amylase activity is considerable. The volume ratio is given when using a protective material with 30% d. see with the enzyme preparation. Since commercial enzyme preparations are generally about 30% d. s., the volume ratios are essentially the same as the weight ratios (based on the dry substance). A preferred one The amount of hydrolyzate materials is generally about 70%, based on the dry substance.

The hydrolyzate material is mixed into the crude bacterial oc-amylase enzyme preparation, and then the mixture is added to a Temperature »heated within the range of about 70 to about 90 C for such a period of time which is sufficient to inactivate the proteolytic enzyme activity without the α-amylase

709886/1012

Significantly reduce enzyme activity. In general, a temperature of about 80 ° C. is preferred. The time during that the temperature is maintained depends on the degree of reduction in proteolytic enzyme activity required and the particular temperature used. In general, periods of 15 minutes to 60 minutes are sufficient although periods of up to 3 hours can also be used. Generally require from Bacillus subtilis Enzyme preparations derived from them have shorter heating times than those derived from Bacillus licheniformis. For example, at 80 ° C., a period of about 15 to about 30 minutes is sufficient for preparations derived from Bacillus subtilis for those derived from Bacillus licheniformis, a period of about 45 to about 60 minutes is required.

The use of the starch hydrolyzate materials as protective materials has the further advantage that practically none Impurities are introduced that must be removed in later stages. The cc-amylase enzyme preparation used for the manufacture of starch hydrolyzate materials is to be used, and the introduction of substantial quantities of the previously prepared hydrolyzate materials does not lead to any subsequent purification problems. In contrast, the Second embodiment of the invention, in which the protective material contains or consists of calcium ions, the calcium ions constitute an impurity which is later removed from the end product must be removed.

In the second preferred embodiment of the invention, calcium ions are used as a protective material. This embodiment is particularly suitable when relatively heat-stable α-amylases, such as. B. those from Bacillus

709886/1012

licheniformis can be used. When using this method, the total amount of Calcium ions are taken into account including those already included in the raw preparation. Since the calcium ion content varies from batch to batch, the calcium ions present must be determined. It can then be a water-soluble one Calcium salt can be added to bring the calcium ion concentration up to a predetermined value. It is essential that the presence of calcium ions tend to increase protease activity as well as α-amylase activity protection. Therefore, a relatively longer heat treatment time may be necessary for higher calcium contents. in order to inactivate the protease enzyme practically completely.

The concentration of calcium ions, which offers the α-amylase activity sufficient protection, is, as has been found, within the range of about 0.5 to about 1.5% enzyme on dry matter basis. The concentration is preferably at least about 1.0%, based on the dry substance. Concentrations of 2.0% calcium ions or more appear to have an adverse influence. In general, the protease content in a Bacillus licheniformis-a-Amylaae, z. B, in Thermamyl, decreased by about 90% by adding that Enzyme as is (with approx. 1.0% Ca) only heat-treated for 60 min at 80 ° C. There is a loss of α-amylase activity observed by only 5%.

The protease-free α-amylase preparations can then be used for Production of starch hydrolysates from a wide variety of starch sources with a high protein content, such as maize, mill, wheat and the like., can be used. This includes both dry and

709886/1012

also wet-milled products, such as corn grits, different types of mill starch, wheat waste and the like. When using the oc-amylase, whose protease activity has been inactivated according to the invention for the hydrolysis of the various starch sources gives hydrolysates that are equivalent in their carbohydrate composition to those made from primary starch will. It is also possible to use oc-amylase enzyme preparations to use that are already devoid of a significant amount of proteolytic activity, e.g. B. the one under the warehouses Maxamyl LX 6000 by the company Gist-Brocades, DeIft, The Netherlands (that of Bacillus subtilis originates). Satisfactory results are obtained using both hydrolysis processes in which gelatinization the strength is avoided, as described in the above-mentioned patents, as well as when using the conventional Enzyme hydrolysis process. For the production of hydrolysates The following enzyme preparations were used from the starch sources:

1.) Thermamyl 60-α-amylase, Charche AN-1009 from Bacillus licheniformis. The original protease activity was 33 084 U / g and the α-amylase activity was 1006 U / g. A volume part Enzyme was added with two volumes of a corn syrup with 30% d.s. and a D.E. diluted by 42. The mixture was heated from room temperature to 80 ° C. over 30 minutes, kept at 80 ° C. for 75 minutes and then in an ice bath cooled down. This resulted in an α-amylase activity of 323 U / g and a protease activity of 520 U / g.

2.) Maxamyl LX 6000-cxTAmylase from Bacillus subtilis with a α-amylase activity of 14,955 U / g. This enzyme instructed

70988 6/1012

Protease / a-amylase ratio was less than 1 and was used as it was.

3.) Aspergillus niger glucoamylase with an activity of 284 glucoamylase-U / g.

The following starch sources were used, with satisfactory ones Results were obtained: 1.) Corn grits / a dry milled corn product, 2.) Corn waste: the fine particles from sieving out corn kernels, 3.) Wheat bran: a dry-milled wheat product, 4.) light mill starch: a starch freed from seedlings

slurry with 10 to 12% protein,

5.) heavy mill starch: a concentrated slurry, 6.) second hydroclone drain: a mill starch slurry, in which the protein is reduced to about 1 to about 1.5%

has been.

The starch materials must be pre-washed prior to introduction into the α-AmyIase enzyme preparation to remove soluble materials, primarily ash and protein; to remove from it. Preferably, the pre-wash is carried out at about room temperature, since at higher temperatures the solubilization of the proteinaceous and carbohydrate materials can be increased. When using dry-milled products such as corn grits or corn bran, the pre-washing is carried out in such a way that 1 part by weight of the starch material is mixed with two parts by weight of water containing about 200 to about 1000, preferably about 500 ppm SO " , slurried for about 1/2 hour at room temperature. The slurry is filtered and washed with at least one more part by weight of the SO ₂ solution. When using wet-milled materials in the form of a slurry

709886/1012

present, which already contains S0 ~, this only needs to be filtered and to be washed with water. Alternatively, these materials can also be washed with the SO_ solution. The grainy one Starch Enzyme Conversion Procedure (GS-EHE) is carried out as follows:

The cake obtained is slurried in water to a solids content of 30% and converted as follows: the slurry is brought to 60 ° C., 50 ppm Ca, based on the dry weight of the starch, is added, the pH is adjusted and 1 U is added PI (inactivated protease) Thermanyl / g ds too. The temperature is raised to 75 ° C at 1 ° C / 5 min up to 70 ° C and then at 1 ° C / 15 min up to 75 ° C. It is placed in a bath with boiling water, left to stand for 15 minutes at 75.degree. C., a heat treatment is carried out at 100.degree. C. and held for 15 minutes. It is cooled to 60 ° C., the pH value is adjusted to 4.3 and 0.14 U glucoamylase / g ds is added or the pH value is adjusted to 5.5 and 0.2% malt extract is added. All final hydrolyzates are adjusted to a pH of 4.3 before filtering. The initial pH adjustment should be about 6.5 when using high protein wet milled materials and about 5.5 when using dry milled materials or starches that have been deproteinized.

Since the gelatinization temperature range for wheat starch is about 10 C lower than that for corn starch, this changes Dilution temperature; urprofile in "a 50 C to 65 C cycle. The Barley malt extract was prepared by stirring 10.38 g of ground malt in 79.26 g of H_0 for a period of 1 hour and then filtering under the influence of gravity through a No. 1 Whatman paper. 10 ml correspond to des

709886/1012

- 2Θ--

Filtrate 1.2 g d. b. Malt. Similarly, the above can be used other granular starch or a granular conventional one Procedures are used.

The protease-free α-amylase preparations can also be granular Starch or conventional processes can be used in which a primary starch is used as the starting material. These starches generally have a protein content of from about 0.30 to about 0.35 percent or less. The use of protease free α-amylase preparations generally lead to hydrolysates with less than about 0.10% protein. When using a primary starch, prewashing is not necessary, it however, it is preferred to achieve the best quality hydrolyzate.

The invention is illustrated in more detail by the following examples, but without being limited to it.

Beisj-el 1

Example 1 illustrates the influence of the concentration and the Type of substrate material on the inactivation of the proteolytic enzyme activity during the heat treatment of an α-amylase enzyme preparation, derived from Bacillus licheniformis. The preparation is mixed with the substrate material and then held at 80 C for the specified period of time. From the data given below it can be seen that the 11 D.E. corn syrup and the 42 D.E. corn syrup against oc-arylase activity Protect from heat inactivation, resulting in products that have a P / a activity ratio within the desired range to have. However, the dextrose does not protect α-amylase activity as well.

709886/1012

With regard to Example 1 and also with regard to the following examples, it is essential that the protease assay in a pH is carried out within the range of about 6.2 to about 6.4 even if the pH is in use possibly lower, for example has a value of about 5.5. However, this has been found necessary since protease assays are not linear with sample size at pH 5.5.

The results of the example are given in Table I below. The heat treatment in the presence of the 11 DE or 42 DE substrate leads to a protease inactivation of about 93% or about 96 % with enzyme / substrate ratios of 1: 1 or 1: 2. A noticeable loss of α-amylase activity is not observed at any ratio. However, when dextrose is used as the protective substrate material, only 89 to 92% protease inactivation is achieved with a decrease in cc-amylase activity of about 12%.

It has now been found that the presence of corn syrup with increasing the heat treatment time from 60 to 75 or to 90 min the protease inactivation increases only slightly, i.e. H. from about 91 to about 95% with no significant loss of α-amylase activity occurs. The reduction in heat treatment time however leads to a considerable decrease in protease inactivation to only 73% after 30 min. that practically the same degree of protease activation was achieved using corn syrup that on a pH was adjusted to 5.5 or 7.0.

709886/1012

Table I.

Influence of the substrate type and concentration on the inactivation of the protease during the heat treatment of Thermarayl ^a 'at 80 ⁰ C

Volume ratio Heat treated
lunß pH Protease activity '
(U / ml) warmth
hands it Protea
be a-amylase ab
activity (U / ml) 585 oc-amy-
read Ρ / α-Ak-
tivit.- Substrate ' Enzyme: Sub
strat Time
(min) 6.4 unconcerned
acts 1,325 active. 391 inact.
(%) Relationship
nis 11 DE
Corn syrup 1: 1 60 6.4 17,080 485 92.2 unheated
acts acts 603 0 2.3 11 DE
Corn syrup 1: 2 60 6.4 11 387 1 190 95.7 591 396 0 1.2 42 DE
Corn syrup 1: 1 60 6.4 17 080 465 93.0 394 522 0; 2.0 42 DE
Corn syrup 1: 2 60 6.3 11,387 1,850 95.9 591 346 0 1.2 Dextrose 1: 1 60 6.2 17 080 870 89.2 394 11. 7 3.5 Dextrose 1: 2 60 11,387 92.4 591 12th 2 2.5 394

c » ^a 'batch AN 1005, 2.01% calcium d. b., P / cc activity ratio «28.9 ex?

Substrate at 30 % D.s., adjusted to pH 5.5 before adding the enzyme, determined at pH 6.3

Example 2

The procedure of Example 1 was repeated using TENASE as an α-amylase enzyme preparation. The heating was carried out with and without the addition of the 42 D.E. corn syrup as protective substrate material. The results obtained are summarized in Table II below.

709886/1012

Table II

Influence of the heat treatment time on the inactivation the protease in TENASE

Heat treatment activity ' (U / g) inactivation (%) Ρ / α-Ak-

^ 'Protease.

TENASE (Charehe LF-2155-C) ^d 'diluted with 2 pbv corn syrup ^C '

untreated 3328 589. - - _{5> 7} ^{80 15} - 230 570 92.8 4.9 0.4

- » ^{80 30} Nil _574 100 _ 2.5 <0.1 S undiluted TENASE (Charche LF 2427-Lp ⁷ ~~~ ~ ~

£ 'untreated ^{9410 1724} "- 5.5 S! 80 15 6967 ND ' 26.0 NDND

O 80 30 3213 904 65.9 55.1 3.6 80 45__ 2051_ 662 78.2 69.4 3.3

^a ^ Activities of the diluted LF-2155-C, determined on a volume basis, calculated on

Weight base using the predetermined specific gravity of 1.15

Protease activity, determined at pH 6.5 for the batch LF-2155-C and at 6.3 for the Lot LF-2427-L

^c ^ 30 w / w% 42 DE corn syrup containing 200 ppm db of added calcium ^ J ^α; 1.55% Ca ⁺⁺ , db cn

^e) 1.94% Ca ⁺⁺ , db g

f)

not determined

Example 3

Samples of THERMAMYL (Batch AN 1050) -a-amylase obtained from on dry weight containing 0.50% calcium were heat treated at 80 ° C for various periods of time. The trials were carried out at both the initial pH of the α-amylase preparation and pH adjusted to 5.5. The results are summarized in Table III below.

The results show that a 60 minute heat treatment at each pH protease / α-amylase activity ratios of less than 2. When the heat treatment time was extended to 120 minutes, a ratio of 0.6 was obtained, but at the expense of 11 to 13% of the α-amylase activity. A slightly higher inactivation of both the protease and dar a-amylase was at a pH of 5.5 as. bei a A pH of 6.4 achieved due to the generally lower enzyme stability in the more acidic medium.

709886/1012

copy i

ORIGINAL INSH = CTBD Table III

Influence of the heat treatment time and the pH value on the inactivation of the protease in THERMAMYL '

Heat treatment Activity (U / g)

Inactivation (%) Ρ / α-

Temp.
( ⁰ C) Time
(min) 30th Protease 528 α-amylase 6.4 Protease α-amylase Activity
relationship 45 517 i.e. 075 untreated 60 960 1, 999 - - 18.2 80 75 4th 980 026 76.9 - 7.1 4.5 80 90 2 598 ' 1 022 84.8 4.6 2.9 80 120 1 152 1 003 89.8 4.9 1.9 80 1, 708 1 964 91.8 6.7 1.6 80 mdelt 1 956 94.1 10.3 1.2 80 45 937 5.5 96.3 11.1 0.7 60 038 EiL 005 unaffected 75 20th , 520 1 992 - - 8/20 ^! 80 90 2 072 920 90.3 8.3 2.2 80 120 1 762 916 92.7 8.5 1.7 80 1 356 896 94.9 8.9 1.2 80 870 96.4 10.8 0.9 80 98.3 13.4 0.4

'Batch AH 1050, 0.50% d. b. Calcium, heat treated in

Absence of carbohydrates

709886/1012

COPY

ORIGINAL INSPECTED

- Yt-

Example 4

Samples from different batches of THERMAMYL were heat treated at 80 C for 60 minutes. Before the heat treatment, the calcium ion content of each batch was determined by adding CaC1 _? set to a predetermined percentage. The remaining protease and α-amylase activities are given in Table IV below. With increasing THERMAMYL calcium content from 0.04% to 2.0%, the protease inactivation decreases from 100% to 91%. In each case the α-amylase inactivation decreases from 25 % with 0.04% calcium to about 5% with 1% calcium. The increasing calcium content to 2 % leads to an increased inactivation of 7% to 16%.

709886/1012

Table IV Influence of the calcium content on the inactivation of protease in THERMAMVL ³ ''

Total
hold Ca ⁺⁺ % ds « Protease activity
(U / g) 178 27 α-amylase activity
(U / h) Inactivation 99.8 α-amylase Ρ / α-active
vity Batch CL db) 35.4 untreated. heat treat. 218 64 untreated. heat treat. Protease 99.6 25.0 Proportions AN 1001 0.04 34.3 16, 173
528 1 597
980 1069 802 96.1
89.8 21.9 0.03 AN 1001 0.25 34.3
26.0 15th 659
746 2 853
229 1031 805 94.6
88.7 10.5
4.9 0.08 AN 1001
AN 1050 0.50
0.50 34.6
29.0 15th
19 899
211 2 869
645 1010
1075 904
1022 94.5
90.9 3.7
5.7 0.66
1.94 4.N 1001
AN 1069 1.00
1.00 33.9
30.4 15th
19th 971
1061 935
1000 7.4
16.5 0.91
2.23 AN 1001
AN 1005 2.00
2.01 15th
29 984
999 911
834 0.95
3.17

Heat treatment was carried out in the absence of carbohydrates for 60 min at 8O ⁰ C.

co I ^

OO CO

o> cn

- · 'oo

- 39- -

Example 5

Various batches of THERMAMYl containing 0.4 to 2.21 % calcium were diluted with 2 parts of 30 % ds-42 DE corn syrup at pH 5.5 and at 80 ° for 30, 60 and 90 minutes C and heat treated for 10, 20 and 30 minutes at 85 ° C. In all cases the pH during the heat treatment was 6.2 to 6.6. The data in Table V below show that

a) the heat treatment of Thermamyl (batch AN 1001) at 80 or 85 C to virtually complete protease inactivation with a loss of α-amylase activity of less than 7% leads,

b) with batch AN 1002 a stronger α-amylase inactivation, however, less protease inactivation is obtained than the other lots of THERMAMYL; the reason for this is yet unknown;

c) the results obtained with batches AN 1005 and 1009 show that (1) treatment at 80 ° C for 90 minutes results in inactivation of the protease of 93 to 94% with a cc-amylase loss of 8 % or less, while (2) a 10 to 30 minute treatment at 85 C results in inactivation of the protease of 97 to 100% with an α-amylase loss of about 7 to about 16%.

709886/1012

27S548Q

Table V Parameters affecting the selective inactivation of proteases in

of THERMAMYL-cc-amylase control heat treatment activity after inactivation (%) P / a-

Templ time heat treatment (u / ml) ratio

(min) Protease ct-Amylas6 Protease ct-Amylase _

C harge. ANlOOl (0.04% db calcium) 30 30 ND 99.6. 0 ^c * 0.1

85
85
85 10
20th
30th 55
0
30th 409
417
444 99.2
100
99.6 7.3
5.4
ϊ 0
■ * o.i l
0.0 ι
0.1 Batch _AN100 2 (0.89% d.b. Calcium) 00 00 00
O O O 30th
60
90 2860
1965
1055 N.D.
N.D.
385 69.8
79.2
88.9 N. D.
N.D.
12.3 4.5 ^b>
2.7 85
85
85 10
20th
30th 1385
405
270 361
341
356 85.3
95.7
97.8 17.8
22.3
18.9 3.8
1.2
0.8 Batch ANl005 (2.01% d.b. Calcium) 80
80
80 30th
60
90 3291 ^C !
⁴⁶⁵ C
774 ^C) N.D.
396
N.D. 71.1
95.9
93.2 0
(T 8.4
1.2
2.0 85
85
85 10
20th
30th 400
0
40 363
342
359 96.5
100
99.6 7.9
13.2
8.9 1.1
0.0
0.1 ChargeAN1009 (2.21% d.b. Calcium) 80
80
80 30th
60
90 3680
1460
475 N.D.
N.D.
369 73.0
89.3
96.5 N.D.
N.D.
8.4 9.1
3.6
1.3 85
85
85 10
20th
30th 430
0
30th 346
337
376 96.8
100
99.8 14.1
16.4
6.7 1.2
0.0
0.1

^a 'Enzyme diluted with 2 pbv 30% 24 DEM corn syrup before heat treatment

it was assumed that no loss of the α-amylase activity of the Samples occurred

'' determined at pH 5.5

709886/1012

Example 6

The protease inactivation process was carried out on a larger scale for the production of 2 large batches of enzyme for conversion studies. The batch sizes were 2600 and 6655 ml THERMAMYL (batch AN 1009) diluted with 2 pbv 42 DE corn syrup with 30% solids and a pH of 5.5. The diluted enzyme (pH 6.2) was heated in 30 minutes in a heated water bath with water vapor at 80 ⁰ C, held for 75 minutes at this temperature and cooled to room temperature within minutes dtwa 30th The final pH was 5.9. The protease inactivation was 93 to 96 % with an α-amylase activity loss of only about 3%. These two batches of heat treated THERMAMYL were used to convert primary starch and mill starch, respectively, to dextrose hydrolysates using the granular starch-enzyme-heat-enzyme (EHE) process.

Example 7

Corn grits (corn scrot) was slurried in water and diluted with THERMAMYL enzyme preparations that are heat-treated with varying degrees of protease inactivation achieved. A specimen was then given to one pH 5.5 was converted to sucrose with glucoamylase from Aspergillus niger and the other was converted with cc-arylase converted to sucrose. The results are summarized in Tables VI and VII below.

709886/1012

Influence of the THERMAMYL protease / α-amylase activity ratio on the production of dextrose hydrolyzate from corn grits

Experiment No. 61 62 63 64 65

ANlOOl AN1005 ANlO09 AN1009 ANlOOl mERMAMYL treatment 80 80 80 80 un ^ ehan- Batch 30th 60 60 30th Temperature ( ⁰ C) 0.1 1.2 3.6 9.1 15.5 Time (min) • P / a activity ratio Hydrolyzate analyzes 29.4 29.5 29.5 29.7 29.7 d.b. 93.8 93.8 94.0 94.0 93.6 % d. s. 0.18 0.22 0.25 0.34 0.48 Dextrose (%) 36 42 57 89 Protein (%) χ 2 16 14th 14th 16 Amino N. (ppm) (oai / s
Filter speed ^N f _t 2)
(Liter / min / m ² ) ^{rc #;} 8th 11 10 10 11

97. 0 97. 2 98 0 98 2 66. 1 66. 4th 66. 7th 66. 9 6th 8th 7th 2 7th 2 7th 1

Residue analyzes, free of soluble substances

Base, d.b.

! EaTaT. 97.0

Protein (%) ⁶⁵ · ⁷ Strength (%) ⁷ - °

Solubilization (% d.b.)

strength
total quantity

^a ', the experiments were for the 75 ⁰ C-OR-BEFORE-Verfären using a 0.05% SO - 5.5 prewash performed, ppm using 50 calcium and conversion to sucrose at pH. the
Dosage of the glucoamylase was 0.14 U / g ds

99 1 99 2 99 .1 99 1 99 1 88 5 88 2 88 .2 88 2 89 1

709886/1012

Table VII

Influence of the Thermamyl protease / α-amylase activity ratio on the production of a syrup with a high maltose / maltotriose content from corn meal (corn grits)

Attempt no.

THERMAMYL treatment

Batch

Temperature (C)

Time (min)

Ρ / α activity ratio

54

55

57

58

ANlOOl AN1005 'AN1009

80 80 80

30 60 30 0.1 1.2 3.6

Hydrolyzate analysis (d.b.)

AN1009 ANlOOIj

80 untreated 30 · »

9.1 15.5

% d.s. 27.9 27.9 27.8 27.8 27.9 D.E. 47 46 46 46 46 DP-I (%) 3 4th 4th 4th 4th DP-2 (%) 53 52 51 53 53 DP-3 (%) 22nd 24 24 24 24 Protein (7o) 0.12 0.16 0.18 0.25 0.43 Amino N. (ppm) ftv) 20th 26th 32 47 83 Filtration speed (gal / Sta. /
Liters / min / m 10 14th
10 16
11 16
11 16
11 Residue analyzes, from soluble fractions free Base, d. b. % ds 96.6 96.6 96.7 97.6 97.3 Protein (%) 63.8 62.9 64.3 63.6 64.4 Strength (%)
soluble parts (%) 11.0
1.3 11.0
2.4 6.9?
1.2 10.0
0.8 9.5
1.2 Solubilization (% d. B.) strength 98.6 98.6 99.1 98.7 98.8 total quantity 88.4 88.6 88.5 88.6 88.9

^a 'the experiments were carried out using the 75 ^° C-GS-EHE method using a 0.2% barley malt extract.

709886/1012

3Γ

Example 8

The influence of protease content on high temperature liquefaction was briefly investigated using THERMAMYL (Lot AN1OO9) which was either heat treated to inactivate the proteases (Ρ / α ratio = 1.4) or "as it was "was used without protease inactivation (P / a ratio = 32.9). 25 w / w% starch slurries, pH 6.2, containing 200 ppm db of added calcium, were inoculated with 2 U / g ds THEBMAMYL. The slurries were held at 50 ° C for 30 minutes or 24 hours prior to liquefaction. The long hold time was used to simulate an extreme case of actual process conditions. Each slurry was diluted by indirect heating for 90 minutes at 90 ° C at pH 6.2 and for 96 hours at pH 4.3 and 60 ° C with 14 glucose araylase U / 100 g / ds in sucrose convicted. The average results obtained in these test series and in a repetition of the test series are given in Table VIII below. The results show that by increasing the holding time from 30 minutes to 24 hours, slightly more soluble protein is obtained in the final hydrolyzates. However, the main factor influencing the hydrolyzate protein content is the protease content in the α-amylase. By using the treated THER ^ MAMYLS (with a low protease content) the protein content is reduced from an average of 0.21% to 0.13% , which corresponds to a reduction of 38%.

709886/1012

3b

Table VIII

Influence of the THERMALYL protease content in an enzyme-enzyme-dextrose process

Attempt no.

28.5 28.5 28.5 95.4 95.4 95.3 0.15 0.20 0.11 1.31 1.31 1.42'i

THERMAMYL untreated treated untreated "treated" j

Protease / oc-Aktiv.-Rat. 32.9 1.4 32.9 1.4 ■

Starch on slacking-hold-24 hours. 24 hours. 30 min. 30 min. Time at 50 ° C ^a;

Liquefaction ¹ ^% ds 26.8 26.8 26.7 26.5

D. E. 22.2 21.9 19.2 19.1

c)

Conversion into sucrose

% ds 28.5

Dextrose (% d.b.) 94.9

Protein (% d.b.) 0.22

insoluble part (% d.b.) 1.16

a 25 w / w% slurry of corn starch containing 2 U / g ds, Thermamyl + 200 ppm db added calcium, was held at 50 ° C. and pH 6.2 for the time indicated before liquefaction

indirect heating to 90 C for a period of 90 min

pH 6.2

^C ^ 60 ° C, pH 4.3, 96 hours, 0.14 GA U / g ds

709886/1012

Example 9

The 75 ⁰ C-GS-EHE method was used to assess maize grits (maize meal) and maize waste. The results obtained are summarized in the following Tables IX and X, For comparison purposes) corn grits according to the 90 C-enzyme-enzyme process umgewandelt.Der meal was mixed with a 500 ppm s0_ solution prewashed, minutes 90 by heating to 90 C liquefied and then converted with glucoamylase or malt extract. The results in Table XI below show that the hydrolyzate composition and quality are the same as obtained from the 75C process. However, the starch solubilization and the filtration rate are higher when using the 75 Ci process, so that the 75 C process is the preferred conversion process.

709886/1012

Table IX

Conversion of corn grits

GEH-32 prewash

Removal of soluble components (% d.b.) 0.05% Conversion into sucrose GA

Hydrolyzate analysis (d.b.)

% ds 29.4

Dextrose 00 94.5 protein (%) 0.33

Amino N. ppm 2 ^ 5

Filtration speed (gal./hours/ft) 5

(Liter / min / m) 3

Residue analysis (d.b.)

% d. see 97.5

Strength (%) 13.8

Protein (%) 67.3 Soluble Ingredients 00 2.0

Solubilization, (d.b.)

Total amount (% T ^ 88.1

Starch (7 ") ^C; 98.2

Conditions. 30 wt / wt% owned _f S0 "washed corn meal,

diluted with 1 U PI THERMAMYL / g ds with 50 ppm Ca, pH 6.5, heated within 2 hours from 60 to 75 ° C, heat-treated at 100 ⁰ C, converted with 0.14 U GA / g dsj pH 4, 3.95 hours at 60 ° C

corrected on a soluble material free basis

contains insoluble materials that were removed during the pre-wash

it was assumed that there was no starch loss in the pre-wash .

709886/1012

Table X
Conversion of maize waste

Pre-washing removed soluble components (% d.b.) Conversion to sucrose

Hydrolyzate analyzes, d. b.

% ds dextrose (%) DE
DP-I (%)

dp-2 cS

Amino N. (ppm) "

Filtration speed (gal./hour/ft) ^lf (liter / min / m)

Residue analysis, d. b.

% d.s.

Starch (%) Protein (%)

soluble components solubilization, db

Total amount ν (%) starch (%) ^c;

a)

GO-21 GO-25 0.05% SO ₂ 0.05% SO- 6.2 6.3 ² Paints X GA 24.2 25.3 91.2 44.1 - 5.9 j - 54.5 - 22.6 - 17.0 - 0.8 2.2 N. D. 360

0.7

98.7 4.7

32.3 1.1

75.8 98.4

0.7

97.9 4.3

30.8 1.2

75.5 98.5

Conditions: 30 wt./wt. % i ge, SO-washed maize waste, diluted with 1 U PI THERMAMYL / g ds "with 50 ppm Ca, pH 6.5, heated within 2 hours from 60 ⁰ C to 75 ° C, heat-treated at 100 ° C, converted with 0.14 U GA / g ds, pH 4.3, 96 hours at 60 ° C (GEH-25) or 0.2% malt extract, pH 5.5, 24 hours at 60 ° C (GEH-24)

corrected on a soluble material free basis

contains the soluble components removed during the pre-wash

it was assumed that there was no starch loss in the pre-wash

709886/1012

GO-59 GEH-92 2735480 GO-10 90 ⁰ C EE GS-MARRIAGE GS-MARRIAGE Paints X Paints X GA - 39 ~ - 40 26.6 28.2 29.6 Table XI N.D. N.D. 90 C enzyme enzyme 94.9 41.0 44.6 N-D. 2.0 2.8 WALK-60 N.D. 53.2 51.6 90 ⁰ C EE N.D. Conversion of corn meal using the 24.7 27.0 GA N.D. Procedure 20.1
0.10 18.6
0.14 HD
0.35 Attempt no. 20th N.D. 28.9 87 procedure 147 N.D. 96.2 47 Conversion to sucrose N.D. Hydrolyzate analyzes, d.b. N.D. Dry matter (%) N.D. Dextrose (%) N.D. D.E. ND
0.37 DP-I (%) 95 DP-2 (%) 102 DP-3 (7o) DP-4 + .m.
Protein (%) Amino N. (ppm) Ca (ppm)

Filtration speed in 4 14

(gal / hr / ftp
(Liter / min / in) <ι 9 3 10

Residue analysis, d.b.

Dry matter ((%) starch (%)

Protein (%>

soluble components (%)

96.5 97.8 96.6 97.2 11.3 9.6 17.6. 9.6 56.1 66.8 57.4 72.9 1.7 1.5 3.2 1.6

Solubilization, d. b.

Total amount, (%) * >> 86.3 88.3 87.0 98.8

Strength (%) ^c > 98.3 98.7 97.5 98.8

Conditions: with 30 wt./wt. % i according to SO-washed corn meal »diluted with 1 U P.I. THERMAMYL / g d.s. with 50 ppm Ca, pH 6.5, heated to 90 ° C for 2 hours, converted at 0.14 U GA / g d.s., 96 hours at 60 C or 0.2% malt extract, 24 hours at 60 C.

corrected on a soluble basis

does not include the soluble components lost in the prewash (with the exception of GEH-92).

it was believed that there was no starch loss in the pre-wash.

709886/1012

Example 10

Heavy mill starch and a starch slurry from which the Protein was partially removed (second hydroclone effluent) were converted using the GS-EHE procedure, about the use of high-strength sources of milled starch Explain protein content. The results obtained are given in Tables XII, XIII and XIV below.

709886/1012

Table XII
Conversion of heavy mill strength

Attempt no.

pH of conversion

in sucrose

Structure dry matter (%)

Hydrolyzate analyzes _f db

Dry matter {%) Dextrose (%) Protein (%)
Amino N, (ppm) Calcium (ppm)

GO-78 80 4th 82 5 79 5 81 83 4th 4.3 33 .3 33 .5 33 .5 5.5 33. 33.1 .3 .1 .1 33.3 I. .3 .1

(gal / h / ft ² ) (liters / min / m ² )

33. 5 33. 0 33. 5 33. 2 33. 0 33. 7th 94. 4th 94. 3 94. 7th 93. 5 93. 5 93. 6th 0. 48 0. 48 0. 49 0. 40 0. 40 0. 41 190 180 205 95 90 95 48 55 38 64 60 60 7th 6th 10 2 .7 7th 5 4th 7th 1 5 5

l)

Residue analysis db dry matter (%) 94.5 starch (%) 9-0

Protein (7th) ⁷¹ x ²

soluble components (%)

95. Ο 96.0 92.6 96.0 95.7 8th. 4th 7.8 10.6 6.5 7.6 72. 8th 72.9 68.6 72.7 72.2 Ι 2 1.5 2.5 0.9 0.8

Solubilization db . Total amount ν (%) "'' starch (%) ^c;

90.4 89.6 90.2 90.0 89.6 90.1 99.0 99.0 99.1 98.8 99.2 99.1

Conditions: 30% w / w washed mill starch, diluted

with 1 U PITHERMAMYL / g ds with 50 ppm Ca ⁺⁺ , pH 6.5, heated within 2 hours from 60 C to 75 C, heat-treated at 100 ° C. Converted with 0.14 U GA / g ds, pH 4.3, 96 hours at 60 ° C or 0.18 U GA / g ds, pH 5.5, 96 hours at 60 ° C.

corrected on a soluble material free basis

does not contain the soluble components removed during the pre-wash

it was believed that there was no starch loss in the pre-wash occurred

70988K / 1012

Table XIII

Dextrose hydrolysates made from the second hydroclone effluent

Attempt no. DU-I DU-2 DO-3 DU-4 DU-5 H-O-Vorwaschen IX IX IX > X 5X Dilute THERMAMYL
1
5.5
50. enzyme
Dose u / g ds
pH value ₊₊
added Ca (ppm db! 1
5.5 P. 1.
1
6.S
50 1
6.5
50 1
5.5
50 Conversion to sucrose 0.14
4.3 GA (ü / g ds)
PH value 0.14
4.3 0.14
4.3 0.14
4.3 0.18
5.5 Hydrolyzate analyzes, d.b. 32.9
94.6
0.16 Dry matter (%)
Dextrose (%)
Protein (%) 33.7
94.8
0.19 33.7
94.4
0.24 55 32.9
65.1
0.22 32.7
95.0
0.13 Amino N. (ppm) 60 85 58 75 20th Ca (ppm d.b.) 60 58 13th 62 68 Filtration speed
holiday / hour / fo 8th 5 9 8th 9 ¹¹ (liters / min / m ² ) 5 3 5 6th

Total amount of insoluble materials (% d.hif ^{2 8 3} ^ ^{1 2 8 3} * ° ^{2 7}

Conditions: 30% Washed second hydroclone effluent dosed and heated within 2 hours to 60 to 75 ° C, heat-treated at ⁰ C IQO converted with GA - 96 hours at 60 C

709886/1012

Table XIV Malt Conversion of the Second Hydroclone Abstrora

Attempt no. LK-106 LK-107 LK-108 GEK-22 30.9 Pre-wash
removed soluble Be
components (% db) "" ^ * "- ' 6X-H ₂ O
0.7 12X-H ₂ O
0.7 0.05% SO "
A.
0.9 43.1 Hydrolyzate analyzes, d. b. i 1.3 Dry matter (%) 31.0 ^Λ 31.0 31.0 54.7
22.8 D.E. (%) 43.4 43.6 42.1 21.2
0.16 j DP-I 00 0.9 0.9 0.8 i DP-2 (%)
DP-3 (%) 57.0
20.8 56.6
18.6 58.0
18.5 DP-4 + (%)
Protein (%) 21.3
0.47 23.9
0.10 22.7
0.09 Solubilization, d. b.

_n . / "Ν 96.9 96.8 96.7 96.8 Total quantity (%)

Conditions: 30% w / w% washed second hydroclone effluent, diluted with 1 PI THERMAMYL U / g ds, pH 6.5, with 50 ppm added calcium, the temperature was increased to 60 to 75 ° C. within 2 hours , WäfcmebehehandIt at 100 C, converted with 0.2% barley starch extract, pH 5.5, 60 ⁰ C, 24 hours.

709886/1012

Example 11

Light mill strength was measured using the 75 ^C-0 GS-EHE-Varfahrens converted by varying different process parameters.

The following Table XV illustrates the influence of the time of conversion to sucrose on the hydrolyzate composition. Table XVI illustrates the effect on the pH of sucrose conversion. Table XVII, Experiment GEH-71, illustrates the influence of the additional addition of α-amylase to the stage of conversion to sucrose. In the experiment GEH-72 MAXAMYL is used as a-amylase instead of THERMAMYL. The test GEH-73 illustrates the heating of the diluted starch to 121 ° C instead of the standard value of 100 ° C. the light mill starch was also converted with a combination of THERMAMYL and glucoamylase at a temperature of 60 ° C. The results are given in Table XVIII.

709886/1012

Table XV

Conversion of light mill strength: influence of the time of conversion in sucrose

~~ "~ dextrose, protein, amino nit,

% d. s.% d.b. % d.b. ppm d.b.

pH of conversion to 4.3 5.5 4.3 5.5 4.3 5.5 4.3 5.5 sucrose

Zei t of UMWA nd lung sucrose in 24 hours

72 hours 96 hours

30.6 30.7 90.2 86. 1 0. 41 0.35 130 80 31.3 30.8 93.9 91. 9 0. 4 * 6 0.38 160 80 30.9 30.8 94.7 93. 6th 0. 47 0.40 170 85 31.2 30.9 94.5 93. 4th 0. 49 0.41 180 85 31.2 30.9 94.5 94. 2 0. 50 0.42 190 100

115 hours

Conditions; 30% w / w% washed ground starch (3X-H ₂ O), diluted with 1 U PI THERMAMYL / g ds with 50 ppm Ca ⁺⁺ , pH 6.5, heated from 60 to 75 within 2 hours ⁰ C, heat treated at 100 ⁰ C. converted 0.14 mi GA / g ds at pH 4.3, 96 hours at 60 ⁰ C or 0.18 U GA / g ds at pH 5.5, 96 rounds at 60 ⁰ C . * £ Experiment No. GEH-74 for pH 4.3, Experiment No. GEH-75 for pH 5.5

Table XVI

Conversion of light mill strength: Influence of the pH value of the Conversion to sucrose

Experiment no. GEH-66

Pre-wash 3X-H-O

removed soluble part «8. 8 Structure dry matter 32.7 pH value of the conversion in Sacch. ⁵ · ⁵

GEH- 70 3X-H_O

9.3 30.5

5.5

GEH- 67 3X-H

8.7 33.1

4.3

GEH-69 3X-H-C

30.5

Hydrolyzate analysis, db dry matter (%)
Dextrose (%)
Protein (%)

Amino N. (ppm)

Filtration Speed (gal / hr / ft *) 4

2 "(liters / min / m) 3

33. 8th 31. 0 34. 0 30th .9 94. 2 93. 5 95. 3 94 .7 0. 29 0. 47 0. 38 0 .53 60 110 160 215 1 4 4th 9 10

Residue analysis, d. b.

a)

Dry substance (%)
Strength (%)
Protein (%)
soluble components (%) 96.9
10.6
70.0
2.3 95.
4th
75.
6th 8th
7th
9
0 97.
11.
67.
2. 8th
6th
9
0 96.5
4.0
76.4
5.4 Solubilization, d.b. Total quantity. (%) &)
Starch (%) ^c; 90.9
98.9 91.
99 9
6th 91.
98 3
8th 92.0
99.6

Conditions: 30% w / w washed mill starch, diluted

with 1 U PITHERMAMYL / g ds with 50 ppm Ca ⁺ *, pH 6.5, heated within 2 hours from 60 C to 75 ° C, heat-treated at 100 ° C, converted with 0.14 U GA / g

ds at pH 4.3, for 96 hours at 60 C or 0.18 U GA / g ds at pH 5.5, for 96 hours at 6O ⁰ C

corrected on a soluble material free basis

does not include the soluble components removed in the prewash

it was believed that the prewash did not remove any starch

709886/1012

-Vh -

Table XVII

Conversion of light mill starch: influence of α-amylase and the heat treatment temperature

Experiment No. GEH-71 ^a ^

pH of conversion to sucrose 5.5

Hydrolyzate analyzes, d. b.

31.0 93.5

0.56 135
69

Dry matter (%)
Dextrose (%)

Protein (%)
Am ^ no N. (ppm)
Ca (ppm)

Filtration speed ig / hour ft *} ⁶

(Liter / min / m ^Z ) ⁴

Residue analysis d.b.

Dry matter (%) ⁹⁵ - ⁸

Starch (%) 3.9

Protein (%) 76.9

soluble components (%) ⁶ ^ ¹

Solubilization, db .

Total amount ^ (%)
Strength (%) ^;

91.9 99.6

GEH-72 ¹ 4.3

30.6
94.9
0.49

200

217

<1

<1

discarded

Il

GEH-73 ^C) 4.3

31.4 90.7

0.62

450

109

95.5

12.1

71.7

6.6

91.5 98.9

0.5 U P.I. THERMAMYL / g d.s., added during conversion in sucrose

diluted with 3 U MAXAMYL / g ds - 200 ppm db Ca ⁺⁺

'ο

min heat treated at 121 ° C

corrected on a soluble material free basis

does not include the soluble components removed in the prewash

it was believed that there was no starch loss in the pre-wash

709886/1012

Conversion of light mill strength using the 60 ° C

g procedure

Experiment no. GEH-76. GO-77

Structure - dry matter (%) 15.2 15.2

Digestion conditions 96 hours at pH 5.5 243td, at pH

Hydroysate analyzes, db *

Dry matter (%) 9.3 10.0

Dextrose (%) 93.7 94.4

Protein (%) 1.5 1.3

Amino N. (ppm) 600.450

Ca ** (ppm) ₂ 33 ³³
Filtration Speed (gal / hr / ft ^) ¹⁸ * ² *

(Liter / min / m ² ) ¹² 14

a) residue analyzes, db

Dry matter (%) 94.2 93.4

Strength (%) 81.7 81.7

Protein (%) 14.4 15.4

Soluble components (%) 0 '°' ²

Solubilization, d. b.

Total amount of starch (%)

Total quantity x 52.7 56.1

^{C) 5} S- ^{7 61 7}

Conditions: 15% washed mill starch converted with U P.I. THERMAMYL / g d.s. - 0.25 U GA / g d.s.

corrected on a basis free of soluble materials

does not include the soluble components removed in the prewash

it was believed that there was no starch loss in the pre-wash

occurred

709886/1012 COPY *

ORIGINAL fNSPECTBD

Example 12

Corn grits (maize grits) were converted using the 75 C-GS-EHE procedure. One batch was prewashed using fresh water and then the second was prewashed using a 0.1% _{SO 2} solution. The results are given in Table XIX below.

709886/1012

- 5Θ-.

S4

Table XIX

Comparison between washing the corn syrup red with H ₃ O and SO ₂

Type of pre-wash

Prewash ^H 2 ° ⁰ ' ^{1% s0} 2

soluble grain components (%) d.b. 2.2 3.4

Hydrolyzate analyzes

% ds

Dextrose (%) db Protein (%) db Amino N (ppm) db

Ca ⁺⁺ (ppm db)

Filtration Speed (gal / hr / ftij)

(Liter / min / m)

a) EPC analyzes '

% ds

Protein (% db) starch (X db) soluble components (% db)

non-converted starch (% db) ^b '1.8 - 1.4

Conditions: ⁰ '30% shot (Grit No. 1, 6/3/74), diluted with

1 U PI THERMAMyiyg ds, pH 6.5 with 200 pgm Ca ⁺⁺ , heated according to a program from 60 C to 75 C, heat-treated at 100 ° C, converted with 0.14 U GA / g ds at pH 4.3, 96 hours at 6O ⁰ C

Values corrected on a soluble material free basis

based on starch recovered in EPC, it was assumed that there was no loss in washing

Dosages based on dry starch substance

709886/1012

29.44 29.50 92.2 95.4 0.25 0.35 53 112 225 173 6.1 14.9 4.3 10.4 95.8 95.5 63.3 62.9 13.1 11.0 2.1 1.7

Example 13

Primary starch was converted to a soluble starch hydrolyzate using the procedure described below:

A 30% w / w aqueous slurry was prepared, the pH was adjusted to 6.2, 300 ppm calcium ions were added, and the slurry was treated with 1.2 U cx-amylase / g ds (MAMAMYL, Ρ / α - 0.9) inoculated. The slurry was heated to 88 ° C and held at that temperature for 2 hours. It was then heated to 127 C and held at that temperature for 15 minutes. The slurry was then cooled to 85 ° C., inoculated with 0.6 U oc-amylase / g ds and held at this temperature for 3 hours. It was then cooled to 60 ° C, adding enough fresh water to reduce the solids content to 25% w / w, the pH was adjusted to 4.3, and the slurry was 0.18 U glucoamylase / g ds inoculated. These conditions were maintained for 95 hours. The hydrolyzate contained 95.9 ° L of dextrose, db, it had a protein content of 0.08 % db, and it contained a total of 1.4% db of insoluble materials.

While the invention has been described above with reference to preferred Embodiments explained in more detail, but it is for those skilled in the art it goes without saying that it is by no means restricted to this, but that it is changed and modified in many ways without departing from the scope of the present invention.

709886/1012

Claims (23)

Claims 1. Method for the selective inactivation of the proteolytic Enzyme activity in a bacterial OL amylase enzyme preparation , characterized in that one (a) introduces a protective material into the preparation, (b) the mixture thus obtained is heated to a temperature within the range from 70 to 90 ° C and (c) maintaining the temperature for a period of time sufficient to render the proteolytic enzyme practical to inactivate completely without the OL amylase activity to decrease significantly. 2. The method according to claim 1, characterized in that the pH of the preparation is within the range of 5 to 8 lies. 3. The method according to claim 1 or 2, characterized in that the bacterial OL amylase from a bacillus microorganism is formed. 4. The method according to claim 3, characterized in that it the Bacillus microorganism is the Bacillus subtilis microorganism. 5. The method according to claim 4, characterized in that one maintaining the temperature for about 30 to about 90 minutes. 6. The method according to claim 4 or 5 »characterized in that that you can use a corn syrup as a protective material 709886/1012 ORIGINAL tNSPECTBD D. E. used by up to 50. 7 · The method according to claim 6, characterized in that a corn syrup with a d. see from 20 to 40% by weight used. 8. The method according to claim 1, characterized in that the protective material in an amount of 0.5 to 2.0 parts used per volume of enzyme preparation. 9. The method according to claim 3 »characterized in that the Bacillus microorganism is used as the bacillus microorganism licheniformis is used. 10. The method according to claim 9, characterized in that a water-soluble calcium salt is used as protective material. 11. The method according to claim 10, characterized in that the water-soluble calcium salt is used in an amount which have a total calcium ion concentration of 0.5 to 1.5%, based on the dry material, results. 12. The method according to any one of claims 9 to 11, characterized in, that the temperature is maintained for 30 minutes to 3 hours. 13. The method according to any one of claims 9 to 12, characterized in that that at least some of the calcium ions were originally present in the enzyme preparation. 14. Process for the enzymatic production of a starch 709886/1012 hydrolyzate with less than 0.5 % soluble protein materials, characterized in that an aqueous slurry of a starch material is contacted with a bacterial OL amylase preparation which is essentially free of proteolytic activity and the slurry is accommodated for a period of time Reaction conditions hold to produce a soluble starch hydrolyzate with any proteinaceous materials remaining in a substantially insoluble state. 15 · The method according to claim 14, characterized in that one uses such as a bacterial OL amylase preparation, produced by the method of claims 1 to 13 has been. 16. The method according to claim 14 or 15 »characterized in that that a raw starch material is used as the starch material, which is prepared before the preparation of the aqueous slurry has been pre-washed. 17. The method according to claim 16, characterized in that it the raw starch material is a dry milled corn starch material and that the pre-wash under Application of the following stages is carried out: (a) Prepare a slurry of the starch material in one containing 200 to 1000 ppm sulfur dioxide aqueous solution, (b) filtering the slurry to form a filter cake and (c) passing another portion of the sulfur dioxide solution through through the filter cake. 709886/1012 18. The method according to any one of claims 14 to 16, characterized in that that the raw starch material is mill starch derived from corn. 19. The method according to any one of claims 14 to 18, characterized in, that the concentration of the cL-amylase preparation 0.1 to 25 units of activity per gram of starch material amounts to. 20. The method according to any one of claims 14 to 19, characterized in that the aqueous slurry 5 to 40
% By weight starch, based on the dry weight. 21. Bacterial <x amylase preparation that is essentially free is of proteolytic enzyme activity, characterized in that it is produced according to the method according to one of the claims 1 to 13 has been produced. 22. Bacterial OL amylase preparation according to claim 21, characterized in that the ratio of proteolytic
Enzyme activity (U / g) to amylolytic activity (U / g) is less than 0.8. 23. Enzymatisehes starch hydrolyzate, characterized in that
that according to the method of one of claims 14 to 20
has been made. 709886/1012